This invention relates generally to microbial metabolism and antimicrobial therapeutic agents. In particular, the invention relates to the bacterial enzyme thioredoxin reductase, to compounds that inhibit this enzyme, and to the use of these compounds as antimicrobial agents, particularly for the therapy of infections caused by Staphylococcus spp.
The thioredoxin system is composed of NADPH, thioredoxin (Trx) and the flavoenzyme thioredoxin reductase (TrxB). Trx reduction by TrxB involves two half-reactions. In the first half-reaction, the FAD prosthetic group of TrxB is reduced by NADPH and electrons are transferred to cysteines present in the active site of TrxB. In the second half-reaction, oxidized Trx is reduced by TrxB. The thioredoxin system serves to transfer reducing equivalents for reductive enzymes such as ribonucleotide reductase, methionine sulfoxide reductase and vitamin K epoxide reductase. It also mediates protein folding and exerts specific redox control of some transcription factors to modulate their binding to DNA.
The thioredoxin system is of particular importance for redox metabolism in some Gram-positive bacteria. In this regard, certain Gram-positive bacteria, such as staphylococci, lack detectable glutathione (GSH) and glutathione reductase (GSR) which together play a key role in maintaining intracellular thiol-disulfide balance. GSH is the predominant thiol produced by aerobic eukaryotes and some Gram-positive bacteria, is believed to protect aerobic organisms from oxygen toxicity, and participates in a multitude of functions. For example, GSH plays a pivotal role in management of oxidative stress and maintenance and regulation of the redox balance. It acts as a cofactor for peroxide and ribonucleotide reductions, and serves in the conjugation and detoxification of foreign substances. Most organisms contain millimolar intracellular concentrations of GSH which, in concert with GSR and glutathione peroxidase, governs the redox status of the cellular environment. Thus, in microorganisms lacking the glutathione system, such as Staphylococcus aureus, the thioredoxin system, which is able to substitute for some of the glutathione-dependent processes, is of utmost importance.
The TrxB component of the thioredoxin system is a FAD-containing enzyme and belongs to a family of pyridine nucleotide-disulfide oxidoreductases. The bacterial enzyme obtained from Streptomyces clavuligerus, is a homodimer of 35 kDa subunits and has a native molecular weight of approximately 70 kDa. Aharonowitz et al. (1993) J. Bacteriol. 175:623-629. Each subunit of TrxB contains NADPH- and FAD-binding domains and includes an oxidoreductase active dithiol in the conserved sequence -CAT/VC-. Since the cysteine residues of TrxB are relatively inaccessible to the substrate thioredoxin, the enzyme appears to undergo a large conformational change during catalysis.
TrxBs from different mammalian species including calf (Holmgren, A. (1997) J. Biol. Chem. 252:4600-4606), rat (Luthman et al. (1982) Biochem. 21:66628-6633), and human (Arscott, et al. (1997) Proc. Natl. Acad. Sci. USA 94:3621-3626), have been purified and biochemically characterized. The rat liver TrxB has been isolated as a 116 kDa homodimer of 58 kDa subunits, and the mass of human placental TrxB estimated to be 160 kDa by gel-filtration chromatography and 130 kDa (two 65 kDa subunits) by sucrose density gradient centrifugation. The size difference between human TrxB and the smaller bacterial TrxBs is primarily due to differences in the dimer-interface domain. The redox-active cysteines of human TrxB are located in the FAD domain with a 4-amino acid bridge linking the two cysteines. The active-site disulfide of bacterial TrxBs, on the other hand, is located within the NADPH domain and a 2-amino acid bridge links the two cysteines. The 3D structure of the human TrxB is likely to be more similar to GSR than to the bacterial TrxB. Thus, primary amino acid sequence alignment of human TrxB with bacterial TrxB sequences reveals just 23-31% identity, whereas alignment of the human TrxB with GSRs of different sources shows 35-44% identity. Arscott, et al. (1997) Proc. Natl. Acad. Sci. USA 94:3621-3626.
Based on the significant differences that exist between the bacterial and the mammalian trxB genes, and the enzymes encoded thereby, the bacterial TrxB provides a potential target for the development of novel antibacterial drugs with a high degree of selectivity.
Antimicrobial agents commonly used to combat bacterial infections generally interfere with one or more critical steps in the metabolism of the bacterium, resulting in growth inhibition or death of the microbe. However, pathogenic microorganisms, including staphylococci, are developing resistance, and in many cases multiple resistances, to existing antimicrobial agents. In this regard, S. aureus is an opportunistic pathogen of increasing medical concern. It can be aggressively invasive, spreading rapidly through soft tissues, directly invading bones and even entering the bloodstream to produce septic shock and disseminated intravascular coagulation. Infections caused by staphylococci generally fall within one of two categories: those related to toxins produced by the bacterium exclusively, including gastroenteritis, toxic shock syndrome, scalded skin syndrome, and the like; and those related to direct invasion and systemic spread of the organism, including dermal infections, bone and joint infections, staphylococcal pneumonia and empyema, meningitis, cerebritis, endocarditis, bacteremia, septic shock, and the like.
These staphylococcal infections have traditionally been treated with xcex2-lactam antibiotics. However, strains of xcex2-lactam antibiotic-resistant staphylococci (BLARS), such as methicillin-resistant S. aureus (MRSA), have developed and become a widespread cause of fatal nosocomial infection. Infections caused by such resistant staphylococci are treated predominantly with xe2x80x9clast resortxe2x80x9d antibiotics such as vancomycin. Since resistance to these antibiotics would essentially exhaust the current therapeutic arsenal, it is essential that new antibacterial agents be identified.
The inventors herein have identified a bacterial thioredoxin reductase (TrxB) from Staphylococcus spp. that catalyzes, in two half-reactions, the specific NADPH-dependent reduction of thioredoxin (Trx), with the concomitant oxidation of NADPH to NADP+. In the first half-reaction, the FAD prosthetic group of TrxB is reduced by NADPH and electrons are transferred to cysteines present in the active site of TrxB. In the second half-reaction, oxidized Trx is reduced by TrxB.
The thioredoxin system provides a significant metabolic function in staphylococci and other Gram-positive bacteria that do not produce glutathione (GSH). The thioredoxin system catalyzes a broad range of protein thiol-disulfide exchange reactions, donates hydrogen for ribonucleotide reductase which is an essential enzyme in DNA synthesis, and is involved in redox regulation of numerous enzyme activities.
Staphylococcal TrxB differs significantly in its function from that of the mammalian enzyme in two important ways. First, it operates in an intracellular environment lacking GSH and GSH-dependent reductases. Second, its subunits are appreciably smaller than the mammalian enzyme and the dimeric enzyme possesses a substrate specificity distinct from its mammalian counterpart. Inhibition of Staphylococcal TrxB activity may cause depletion of reduced low molecular weight thiols, increase protein thiol oxidation, and interfere with DNA synthesis and radical scavenging. Such compromised cells are more likely to succumb to environmental challenges, such as those posed by the host immune system.
Consequently, bacterial TrxB provides an excellent target for the development of novel antibacterial drugs with a high degree of selectivity.
Such antibacterials act by inhibiting TrxB, thereby incapacitating the target bacterium, with few or no side-effects to the eukaryotic host organism. As a result, inhibitors of TrxB activity are effective antimicrobial agents against Staphylococcus and other microorganisms that depend on TrxB for their redox reactions.
Accordingly, in one embodiment, the invention is directed to an isolated Staphylococcus TrxB polypeptide.
In another embodiment, the invention is directed to an isolated polynucleotide that encodes a Staphylococcus TrxB polypeptide.
In yet other embodiments, the invention is directed to a recombinant vector comprising the polynucleotide molecule, recombinant host cells transformed with the vector, and methods of producing recombinant polypeptides using the transformed cells.
In still a further embodiment, the invention is directed to an oligonucleotide probe capable of specifically hybridizing to a nucleic acid molecule encoding a Staphylococcus TrxB. The probe includes about 8 to about 50 contiguous nucleotides of the TrxB-encoding polynucleotide.
In still another embodiment of the invention, antibodies to a Staphylococcus TrxB polypeptide are provided.
In another embodiment, the invention is directed to a method of detecting the presence of Staphylococcus in a test sample suspected of containing a Staphylococcus, comprising:
(a) contacting the test sample with the oligonucleotide probe above, under conditions which allow a Staphylococcus polynucleotide, when present in the test sample, to hybridize with the oligonucleotide probe to form a hybrid complex; and
(b) detecting the presence or absence of the hybrid complex as an indication of the presence of a Staphylococcus in the test sample.
In yet another embodiment, the invention is directed to a method of detecting the presence of Staphylococcus in a test sample suspected of containing Stapylococcus, comprising:
(a) contacting the test sample with a TrxB antibody, under conditions which allow a Staphylococcus TrxB, when present in the test sample, to bind to the antibody to form an antibody-TrxB complex; and
(b) detecting the presence or absence of the antibody-TrxB complex, as an indication of the presence of Staphylococcus in the test sample.
In another embodiment, the invention is directed to a method of detecting a Staphylococcus in a test sample suspected of containing a Staphylococcus, comprising:
(a) incubating the test sample with a disulfide-containing substrate in the presence of thioredoxin and NADPH, under conditions that favor reduction of the disulfide-containing substrate, to produce a detectable signal when a Staphylococcus thioredoxin reductase is present;
(b) detecting the presence or absence of the signal; and
(c) correlating the presence of the signal with the presence of Staphylococcus in the test sample.
In yet further embodiments, the invention is directed to diagnostic kits comprising an oligonucleotide probe as above, a TrxB antibody, or a Staphylococcus TrxB polypeptide, and instructions for conducting the diagnostic test.
In another embodiment, the invention is directed to a method for identifying a compound that modulates Staphylococcus TrxB activity, comprising:
(a) providing a Staphylococcus TrxB polypeptide capable of catalyzing the specific reduction of thioredoxin with the concomitant oxidation of NADPH to NADP+;
(b) contacting a test compound with the TrxB polypeptide in the presence of thioredoxin, NADPH and a disulfide-containing substrate, under conditions that favor reduction of the disulfide-containing substrate;
(c) monitoring the presence of free sulfhydryl groups formed by the reduction of disulfides of the disulfide-containing substrate, as a measure of TrxB activity,
thereby identifying a compound that modulates Staphylococcus TrxB activity.
In yet further embodiments, the invention is directed to compounds identified by this method, compositions comprising the compounds and methods of treating Staphylococcus infections in an infected subject, comprising administering effective antibacterial amounts of the compositions.
In another embodiment, the invention is directed to a method for isolating a TrxB polypeptide from a Staphylococcus cell culture extract, comprising:
(a) performing a protein precipitation step with the cell culture extract to yield a first TrxB mixture;
(b) subjecting the first TrxB mixture to gel-filtration chromatography;
(c) identifying fractions from step (b) with TrxB activity; and
(d) performing anion-exchange chromatography on the fractions with TrxB activity to yield a product with a greater concentration of TrxB than the first TrxB mixture.
These and other embodiments of the present invention will readily occur to those of ordinary skill in the art in view of the disclosure herein.